Project summary The rapid detection of Mycobacterium tuberculosis in clinical samples with a low bacterial load is a challenge to the diagnostic laboratory. Nucleic acid-based approaches such as PCR offer much promise in this area owing to their speed and their high sensitivity. devR- and hupB-based PCR assays were developed in the Indian Pis' laboratories that show high diagnostic accuracy in pulmonary and extrapulmonary tuberculosis disease settings in human and bovine samples from subjects suspected to suffer from tuberculosis. The hupB assay distinguishes M. bovis from M. tuberculosis both of which are closely related members of the MTB complex. In this project it is proposed to optimise molecular beacon and / primer design for the reliable detection ofdevR and hupB PCR products in a visual format (in US Pi's laboratory). Combined with the robust Universal Sample Processing (USP) technology developed by Indian PI (Jaya S. Tyagi), PCR is poised to become a user-friendly test for diagnosing tuberculosis in all types of clinical samples. The standardized assays will be tested in the Indian Pi's laboratories on clinical samples obtained from suspected human tuberculosis patients (pulmonary & extra-pulmonary tuberculosis) and from farm animals processed by USP technology. It is expected that the results of this study will establish universally applicable, robust, user-friendly and high throughput diagnostic and / screening assays for identification of Mycobacterial pathogens of the MTB complex prevalent in samples of human and bovine origin.